FP 2014 46RR.Pdf

UNIVERSITI PUTRA MALAYSIA

PERFORMANCE OF PROBIOTIC Bacillus subtilis G1 AS A DIETARY SUPPLEMENT FOR Hemibagrus nemurus Valenciennes FINGERLINGS

FARHANA AHMAD AFFANDI

FP 2014 46

PERFORMANCE OF PROBIOTIC Bacillus subtilis G1 AS A DIETARY SUPPLEMENT FOR Hemibagrus nemurus Valenciennes FINGERLINGS

UPM

FARHANA AHMAD AFFANDI

October 2014 All material contained within the thesis, including without limitation text, logos, icons, photographs and all other artwork, is copyright material of Universiti Putra Malaysia unless otherwise stated. Use may be made of any material contained within the thesis for non-commercial purposes from the copyright holder. Commercial use of material may only be made with the express, prior, written permission of Universiti Putra Malaysia.

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PERFORMANCE OF PROBIOTIC Bacillus subtilis G1 AS A DIETARY SUPPLEMENT FOR Hemibagrus nemurus Valenciennes FINGERLINGS

By FARHANA AHMAD AFFANDI October 2014

Chairman: Associate Professor Che Roos Saad, PhD Faculty: Agriculture

A study was carried out to investigate the probiotic activity of BacillusUPM subtilis G1 isolated from fermented pickles on Hemibagrus nemurus fingerlings at Universiti Putra Malaysia. Hemibagrus nemurus is a highly price fish that is commercially cultured in Southeast Asia for its excellent taste. However, H. nemurus is a slow- growing fish that take almost a year to reach marketable price. Large-scales aquaculture of this fish has exposed the fish to a stressful condition such as diseases. Aeromonas hydrophila is known to be the famous bacterial infections in Bagridae catfishes. Growth hormones and antibiotics have been used for a long time in aquaculture to increase production and to prevent diseases. However, these substances have caused many problems. Therefore, probiotics are used as an alternative method for sustainable aquaculture.

This probiotic was mixed in feed at doses of 0 (C, control), 109 (T1), 107 (T2) and 105 (T3) cfu g-1 and administrated to H. nemurus fingerlings for nine weeks. Results showed that H. nemurus fed with diet containing 107 cfu g-1 of B. subtilis G1 had significantly higher weight gain, total length and specific growth rate with 248.69 ± 3.31%, 13.65 ± 0.09 cm and 1.98 ± 0.09% respectively, and better protein efficiency ratio and food conversion ratio with 19.71 ± 0.33 and 1.68 ± 0.03 respectively, than those fed with the control, 105, and 109 cfu g-1 diets. Total protein gain of the body fish fed probiotic diets also increases with the increasing weight gain. This probiotic was also found to improve the water quality by lowering the NH3-N concentration. The haematological parameters such red blood cells, haemoglobin, haematocrit and meanCOPYRIGHT corpuscular haemoglobin concentration, in the fish fed probiotic diets was higher compared to the control.

©Inhibitory activity of the probiotic B. subtilis G1 against fish pathogens such as Aeromonas hydrophila and Streptococcus agalactiae was evaluated by well diffusion agar method. Inhibition zone measured showed strong inhibitory activity against A. hydrophila and S. agalactiae with 16.13 ± 0.91 mm and 17.5 ± 1.84 mm respectively. Later, H. nemurus of all groups were fed with their respective diets for three weeks and then were challenged with 106 cfu ml-1 of A. hydrophila (0.1 ml) by intraperitoneal injection. After 14 days, the cumulative mortality of H. nemurus were i significantly lower in the group T1 with 30 ± 5.8% as compared to the T2 (36.7 ± 3.3%), T3 (46.7 ± 3.3%) and C (56.7 ± 3.3%) groups. These findings proved that supplementation of B. subtilis G1 in the diet at 109 cfu g-1 can improve growth and disease resistance in H. nemurus fingerlings.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

PRESTASI PROBIOTIK Bacillus subtilis G1 SEBAGAI PENAMBAHAN DIET UNTUK JEJARI BAUNG (Hemibagrus nemurus Valenciennes)

Oleh FARHANA AHMAD AFFANDI Oktober 2014

Pengerusi: Profesor Madya Che Roos Saad, PhD Fakulti: Pertanian

Satu kajian telah dijalankan uuntuk mengkaji aktiviti probiotik BacillusUPM subtilis G1 yang diasingkan dari fermentasi jeruk ke atas jejari Baung di Universiti Putra Malaysia. Ikan Baung merupakan ikan mahal yang diternak secara komersil di Asia Tenggara kerana rasanya yang lazat. Namun, Baung adalah ikan yang lambat membesar yang mengambil masa hampir setahun untuk mencapai saiz pasaran. Kepesatan penternakan ikan Baung ini telah mendedahkan ikan kepada penyakit. Penyakit dari bakteria Aeromonas hydrophila terkenal sebagai patogen yang menyerang ikan dari famili Bagridae. Pelbagai hormon tumbesaran dan antibiotik telah digunakan sekian lamanya untuk melawan penyakit di dalam akuakultur. Namun, bahan-bahan ini telah menyababkan pelbagai masalah. Oleh itu, probiotik digunakan sebagai satu cara terbaik untuk akuakultur yang mampan.

Probiotik dicampurkan ke dalam makanan ikan dengan dos 0 (C), 109 (T1), 107 (T2) dan 105 (T3) sel g-1 dan diberi makan kepada jejari Baung selama sembilan minggu. Keputusan telah menunjukkan bahawa Baung yang diberi diet yang mengandungi 107 sel g-1 B. subtilis G1 mempunyai peratus pertambahan berat, panjang dan kadar pertumbuhan spesifik yang nyata sekali lebih tinggi dengan nilai masing-masing 248.69 ± 3.31%, 13.65 ± 0.09 sm dan 1.98 ± 0.09%, serta mempunyai nisbah kecekapan protein dan nisbah penukaran makanan yang baik dengan nilai masing-masing 19.71 ± 0.33 dan 1.68 ± 0.03, berbanding dengan diet kawalan, 105, dan 109 sel g-1. Jumlah protein terkumpul dalam Baung yang diberi diet berprobiotik juga bertambah dengan pertambahan berat badannya. Probiotik tersebutCOPYRIGHT juga didapati mampu meningkatkan kualiti air dengan merendahkan kepekatan NH3-N. Parameter hematologi seperti sel darah merah, hemoglobin, hematokrit dan kepekatan min korpuskular hemoglobin Baung yang memakan diet ©berprobiotik turut bertambah berbanding dengan kawalan.

Akitiviti perencatan probiotik B. subtilis G1 terhadap patogen Aeromonas hydrophila dan Streptococcus agalactiae dinilai melalui ujian resapan gel agar. Zon perencatan yang terbentuk menunjukkan aktiviti perencatan yang kuat terhadap A. hydrophila dan S. agalactiae dengan masing-masing 16.13 ± 0.91 mm dan 17.5 ± 1.84 mm. iii

Jejari Baung dari semua kumpulan kemudiannya diberi diet masing-masing selama tiga minggu dan kemudian diuji dengan 106 sel ml-1 A. hydrophila (0.1 ml) melalui suntikan intraperitoneal. Selepas 14 hari, kematian terkumpul Baung nyata sekali lebih rendah dalam kumpulan T1 dengan 30 ± 5.8% berbanding dengan kumpulan T2 (36.7 ± 3.3%), T3 (46.7 ± 3.3%) dan C (56.7 ± 3.3%). Penemuan ini membuktikan bahawa penambahan B. subtilis G1 pada dos 109 sel g-1 dalam diet ikan mampu meningkatkan pertumbuhan dan ketahanan penyakit dalam jejari ikan Baung.

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ACKNOWLEDGEMENTS

Alhamdulillah to Allah S.W.T, for His bless and kindness to me for finishing my master studies. First, I want to thank my main supervisor, Assoc. Prof. Dr. Che Roos Saad for the opportunity to be under his guidance and patiently continuing to encourage me. I am also thankful to my co-supervisors, Assoc. Prof. Dr. Hassan Mohd. Daud and Prof. Mohd. Salleh Kamarudin, for their suggestions and guidance during my study. I am also grateful for their willingness to be part in the committee members, provide me assistance whenever required and involve in reviewing this dissertation.

I also want to thank all staffs in the Aquaculture Department, Faculty of Agriculture for their support and help during the research and studies, especially to Mr. Jasni, Mdm. Nur Shafika, and Miss Norazlina. Not forgotten to the staffsUPM in Aquatic Laboratory, Faculty of Veterinary for their guidance and for allowing me to use the lab and equipment. Many thanks also to my senior Hadi Zokaeifar, for the guidance, and to all my friends for the support.

Finally, I dedicate this thesis to my family, especially my mom and dad, for the faith and support that have given me the inspiration and patience necessary to see this journey through. For that, I am immeasurably grateful.

I certify that a Thesis Examination Committee has met on 31 October 2014 to conduct the final examination of Farhana Ahmad Affandi on her thesis entitled "Performance of probiotic Bacillus subtilis G1 as a dietary supplement for Hemibagrus nemurus Valenciennes fingerlings" in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded the Master of Science.

Members of the Thesis Examination Committee were as follows:

S. M. Nurul Amin, PhD Faculty of Agriculture Universiti Putra Malaysia (Chairman)

Annie Christianus, PhD Associate Professor Faculty of Agriculture UPM Universiti Putra Malaysia (Internal Examiner)

Abdul Razak Alimon, PhD Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner)

Ahmed Jalal Khan Chowdhury, PhD Professor Kulliyyah of Science International Islamic University Malaysia Malaysia (External Examiner)

______COPYRIGHTZULKARNAIN ZAINAL, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia ©

Date: 12 March 2015

This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows:

Che Roos Saad, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman)

Hassan Mohd. Daud, PhD Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member)

Mohd. Salleh Kamarudin, PhD Professor UPM Faculty of Agriculture Universiti Putra Malaysia (Member)

______BUJANG BIN KIM HUAT, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia

vii

Declaration by Graduate Student

I hereby confirm that: • this thesis is my original work; • quotations, illustrations and citations have been duly referenced; • this thesis has not been submitted previously or concurrently for any other degree at any other institutions; • intellectual property from the thesis and copyright of thesis are fully-owned by Universiti Putra Malaysia, as according to the Universiti Putra Malaysia (Research) Rules 2012; • written permission must be obtained from supervisor and the office of Deputy Vice-Chancellor (Research and Innovation) before thesis is published (in the form of written, printed or in electronic form) including books, journals, modules, proceedings, popular writings, seminar papers, manuscripts, posters, reports, lecture notes, learning modules or any other materials as stated in the Universiti Putra Malaysia (Research) Rules 2012; UPM • there is no plagiarism or data falsification/fabrication in the thesis, and scholarly integrity is upheld as according to the Universiti Putra Malaysia (Graduate Studies) Rules 2003 (Revision 2012-2013) and the Universiti Putra Malaysia (Research) Rules 2012. The thesis has undergone plagiarism detection software.

Signature: ______Date: ______

Name and Matric No.: FARHANA AHMAD AFFANDI GS28831

viii

Declaration by Members of Supervisory Committee

This is to confirm that: • the research conducted and the writing of this thesis was under our supervision; • supervision responsibilities as stated in the Universiti Putra Malaysia (Graduate Studies) Rules 2003 (Revision 2012-2013) are adhered to.

Signature: ______Signature: ______Name of Name of Chairman of Member of Supervisory Supervisory Committee: ______Committee: ______UPM

Signature: ______

Name of

Member of

Supervisory

Committee: ______

TABLE OF CONTENTS

Page ABSTRACT i ABSTRAK iii ACKNOWLEDGEMENTS v APPROVAL vi DECLARATION viii LIST OF TABLES xii LIST OF FIGURES xiii LIST OF ABBREVIATIONS xv

CHAPTER

1 GENERAL INTRODUCTION 1.1 Background of Study 1 1.2 Problem Statement UPM2 1.3 Objectives 3

2 LITERATURE REVIEW 2.1 Status of Aquaculture 5 2.2 Classification of Hemibagrus nemurus 8 2.3 Biological Features 8 2.4 Habitat and Biology 8 2.5 Distribution and Economic Status 9 2.6 Catfish Culture 9 2.7 Catfish Pathogen, Aeromonas hydrophila 10 2.8 Probiotics 2.8.1 Definition of Probiotic 12 2.8.2 Selection of Probiotic Bacteria 12 2.8.3 Mode of Action 13 2.8.4 Probiotic Studies in Aquaculture 15 2.8.5 Probiotic Bacteria Bacillus subtilis 19

3 GENERAL METHODOLOGY 3.1 Bacterial Strain 21 3.2 Diet Preparation 21 3.3 Water Quality Analysis 21 3.4 Haematological Analysis 22 3.5 COPYRIGHTHistopathological Assessment 22 3.6 Statistical Analysis 24

4 EFFECTS OF Bacillus subtilis G1 ON GROWTH © PERFORMANCE AND BODY COMPOSITION OF Hemibagrus nemurus FINGERLINGS 4.1 Introduction 25 4.2 Methodology 4.2.1 Bacterial Strain 26 4.2.2 Pathogenicity of Probiotic to H. nemurus 26 x

4.2.3 Feeding Trial 26 4.3 Results 4.3.1 Pathogenicity of Probiotic to H. nemurus 31 4.3.2 Growth Parameters of H. nemurus 33 4.3.3 Body Composition of H. nemurus 34 4.3.4 Water Parameters 35 4.3.5 Haematological Parameters 36 4.3.6 Histopathology 37 4.4 Discussion 40 4.5 Conclusion 43

5 RESISTANCE OF PROBIOTIC FED Hemibagrus nemurus FINGERLINGS TO Aeromonas hydrophila INFECTION 5.1 Introduction 45 5.2 Methodology 5.2.1 Bacterial Strain 46 5.2.2 Antagonistic Activity and Dilution Effect of Probiotic by Agar Well Diffusion Method UPM46 5.2.3 Medial Lethal Dose (LD50) of A. hydrophila Infection 46 5.2.4 Challenge Test 47 5.3 Results 5.3.1 Antagonistic Activity and Dilution Effect of Probiotic 49 5.3.2 LD50 Test 49 5.3.3 Challenge Test 52 5.3.4 Water Parameters 55 5.3.6 Haematological Parameters 55 5.3.7 Histopathology 56 5.4 Discussion 61 5.5 Conclusion 63

6 GENERAL DISCUSSION, CONCLUSION AND RECOMMENDATION 65

LIST OF TABLES

Table Page

2.1 World capture fisheries and aquaculture production and 6 utilization (million tonnes) (Source: FAO, 2012)

2.2 Malaysian capture fisheries and aquaculture production 7 (Source: DoF, 2013)

4.1 Proximate composition (means ± SE) of commercial feed 27 (CP)

4.2 Cumulative mortality of H. nemurus fingerlings at 14 days of 32 post-challenge with probiotic B. subtilis UPM

4.3 Effect of B. subtilis G1 on growth performance of H. 34 nemurus fingerlings

4.4 Proximate composition of H. nemurus whole body tissues (% 35 DM basis) before (initial) and after fed with different diets

4.5 Water quality in the H. nemurus rearing tanks 36

4.6 Haematological parameters of H. nemurus after 9 weeks of 37 probiotic feeding

5.1 Inhibitory activity of B. subtilis G1 supernatant against 49 pathogens

5.2 Means of cumulative mortality of H. nemurus fingerlings 53 challenged with A. hydrophila at 14 days of LD50 test

5.3 Cumulative mortality of H. nemurus fingerlings fed with 54 probiotic at 14 days of post-infection with A. hydrophila

5.4 Water quality during the challenge test 55 COPYRIGHT 5.5 Haematological parameters of H. nemurus fed probiotic diets 56 after 14 days of challenge with A. hydrophila ©

xii

LIST OF FIGURES

Figure Page

3.1 Flowchart of histological procedures 22-23

4.1 Cumulative mortality of H. nemurus fingerlings at 14 days 31 of post-challenge with different doses of probiotic B. subtilis

4.2 (a) H. nemurus fed with probiotic diet is bigger than (b) H. 33 nemurus fed with control diet at 9 weeks of post-feeding

4.3 Spleen of control fish (9-wkpf) showing red pulp area (R) 38 and vacuolative changes (arrow) within the white pulp area (W). Mag. 200x; H&E. UPM

4.4 Spleen of T1 fish (9-wkpf) showing healthy spleen 38 parenchyma with white pulp (W) and red pulp (R) areas. Mag. 200x; H&E; T1, diet + 109 cfu g-1 B. subtilis G1.

4.5 Spleen of T2 fish (9-wkpf) showing increased size of white 39 pulp area (W) and red pulp area (R) within the spleen parenchyma. Mag. 200x; H&E; T2, diet + 107 cfu g-1 B. subtilis G1.

4.6 Spleen of T3 fish (9-wkpf) showing mild atrophy of white 39 pulp (W) and red pulp (R) areas. Mag. 200x; H&E; T3, diet + 105 cfu g-1 B. subtilis G1.

5.1 Infected H. nemurus showing hyperaemic skin 50 discolouration on lateral side of the body

5.2 Pale coloured liver (L) and swollen intestines (I) at 14 days 50 of post-infection

5.3 Yellow mucus-like fluid in the intestine (arrow) 51

5.4 Cumulative mortality of H. nemurus fingerlings fed with 52 COPYRIGHT 6 probiotic at 14-dpi with pathogen A. hydrophila (3.16 x 10 cfu ml-1). C, control (without B. subtilis G1); T1, fed diet + 109 cfu g-1 B. subtilis G1; T2, fed diet + 107 cfu g-1 B. © subtilis G1; T3, fed diet + 105 cfu g-1 B. subtilis G1.

5.5 Liver of control fish (3-wkpf) showing vacuolation of 57 hepatocyte cytoplasms (arrowhead) and hepatic blood vessel containing increase presence of mononucleated WBCs (arrow). Mag. 200x; H&E.

xiii

5.6 Liver of T1 fish (3-wkpf) showing a central vein (arrow) 57 and normal hepatocytes architecture. Mag. 200x; H&E; T1, diet + 109 cfu g-1 B. subtilis G1.

5.7 Liver of T1 fish (14-dpi) showing hepatocyte undergoing 58 degeneration (loss of cell architecture) and hemorrhaging (arrow) in fish challenged with live A. hydrophila. Mag. 200x; H&E; T1, diet + 109 cfu g-1 B. subtilis G1.

5.8 Spleen of control fish (14-dpi) showing increased 59 accumulation of blood in the spleen parenchyma and increased hemosiderin deposition (arrow). Mag. 200x; H&E.

5.9 Degenerated spleen parenchyma of T1 fish (14-dpi) with 59 marked vacuolation (arrow) within the white pulp area (W) and reduced red pulp area (R). Mag. 200x; H&E; T1, diet + UPM 109 cfu g-1 B. subtilis G1.

5.10 Spleen of T2 fish (14-dpi) showing increased deposition of 60 hemosiderin (arrow). Mag. 200x; H&E; T2, diet + 107 cfu g- 1 B. subtilis G1.

5.11 A section from T3 fish (14-dpi) showing degenerated white 60 pulp (W) and red pulp (R) areas of the spleen parenchyma with marked vacuolisation (arrow). Mag. 200x; H&E; T3, diet + 105 cfu g-1 B. subtilis G1.

xiv

LIST OF ABBREVIATIONS

ANOVA Analysis of variance

cfu Colony forming unit

CP Charoen Pokphand

DM Dry-matter

DO Dissolved oxygen

DoF Department of Fisheries

dpi Days of post-infection EDTA Ethylene-diamine-tetra-acetic acid UPM FAO Food Agriculture Organization

FCR Feed conversion ratio

g Gram

h Hour

Hb Haemoglobin

HCl Hydrochloric acid

Hct Haematocrit

K2SO4 Potassium sulfate

L Liter

m Meter

MCHC Mean corpuscular haemoglobin concentration MCVCOPYRIGHT Mean corpuscular volume min Minute ©ml Milliliter mm Millimeter

n Number of samples

NaOH Sodium hydroxide

NH3-N Ammonia-Nitrogen nm Nanometer

NSS Normal saline solution

PER Protein efficiency ratio ppm Part per million ppt Part per thousand

RBC Red blood cell rpm Round per minute

SE Standard error SGR Specific growth rate UPM SPSS Statistical Package for Social Science

TSA Tryptic Soy Agar

TSB Tryptic Soy Broth

UPM Universiti Putra Malaysia

WBC White blood cell wkpf Weeks of post-feeding

xvi

CHAPTER 1

GENERAL INTRODUCTION

1.1 Background of Study

Hemibagrus nemurus is commonly known as the Asian redtail catfish or locally known as Baung (Rainboth, 1996). This catfish is widely cultured in floating net cage along the river rather than in a pond. Hemibagrus nemurus is a highly price aquarium fish but is commercially cultured as live food fish trade for its high nutrient content and good taste. Hemibagrus nemurus is a slow-growing fish that can take almost a year to reach marketable price as compared to other catfishes, under similar culture condition. UPM

Aquaculture is widely practiced in many countries and has become an important source of income. Unfortunately, the large-scales of aquaculture practices have exposed the cultured fish and shellfish to stressful conditions, such as diseases and environmental changes that results to economic losses. Consequently, the fish production is maximised through application of commercial diets, addition of antibiotics, growth promoters, and several other additives. These drawbacks can be overcome by the use of probiotics or beneficial bacteria in aquaculture to control diseases as well as increase growth to reach marketable size in a shorter culture period. Probiotics have directly become an alternative way to antibiotic treatment, winning itself as being more environmental friendly for a sustainable aquaculture (Cruz et al., 2012; Mohapatra et al., 2012).

According to Soccol et al. (2010), probiotics are microorganisms that are capable of living successfully in the human and animal gut, and producing beneficial physiological effects by assisting in the establishment of intestinal microbial population. This will then have an antagonistic effect towards harmful bacteria, thus being advantageous towards the host. The global market of probiotic ingredients, foods and supplements for humans and animals use in 2008 was approximately US$15,900COPYRIGHT million and is estimated to increase up to US$19,600 million in 2013.

As evidence above, probiotics beneficially affect the host by producing inhibitory ©compounds , competing for adhesion site, nutrient and energy source, as source of nutrients and enzymatic contribution to digestion, enhancing immune response, improving water quality, interacting with phytoplankton, and showing antiviral activity (Cruz et al., 2012; Mohapatra et al., 2012; Chiu et al., 2010; Sun et al., 2010; Son et al., 2009; Sahu et al., 2008; Verschuere et al., 2000).

A fast and simple method of culturing, storing and administrating are more preferred by the aquaculturist. Currently, there are several commercial probiotic products that contain one or more live microorganisms that are marketed in powder or liquid forms. Probiotics are applied to cultured species as a feed additive that is either directly apply to water culture, or mixed with feed and administrated orally.

1.2 Problem Statement

Problems in aquaculture are associated with diseases, floods, predation, chemical poisoning, theft, and other miscellaneous causes. However, disease problems have become the most significant factor to aquaculture losses (De et al., 2014; FAO, 2012; Bostock et al., 2010; Meyer, 1991). In recent years, disease outbreaks have affected farms and hatcheries in several countries in Asia, South America and Africa,UPM resulting in a huge loss of production. In 2010, China suffered losses of 1.7 million tonnes of aquaculture production caused by natural disasters, diseases and pollution. In 2011, Mozambique suffered outbreak of disease on their marine shrimp farming (FAO, 2012). Unfavorable conditions such as overcrowding, temperature fluctuations, inadequate dissolved oxygen (Wedemeyer et al., 1976 as cited in Meyer, 1991), and poor culture practices such mishandling, uncontrolled feed usage and untreated waste management (Subasinghe, 2005), may alter the water culture quality thus contributing to an outbreak of diseases.

Growth and survival of catfish fry to fingerlings vary greatly depending on the condition of the culture tank, stocking densities, food abundance and the incidence of infectious diseases. About 45% of inventory losses on catfish fingerling farms are due to infectious diseases. Of the overall losses, 60% resulted from bacterial infections, 30% from parasitic infestation, 9% from fungal infections, and 1% are viral infections (Anonymous, 2010; Al-Dohail et al., 2009). Pathogens from the genus Aeromonas were commonly found to infect the freshwater fishes in Malaysia and of these pathogens were Aeromonas hydrophila (69.6%), Aeromonas caviae (8.7%) and Aeromonas sobria (21.7%) (Anonymous, 2004).

Controls on manyCOPYRIGHT serious infectious diseases have been done through chemical disinfectants, antibiotics and vaccines. However, the use of chemical disinfectants kills not just the bacteria which caused problems to the aquatic species, but also most of the beneficial bacteria in the water column (Sahu et al., 2008). Meanwhile, misuse of ©antibiotic s and the use of unlicensed antibiotics could cause an outburst of antibiotic resistant bacteria (Hashim, 2008; Subasinghe, 2005). Taufik and Bastiawan (2003) have studied the susceptibility of A. hydrophila isolated from Baung to antibiotics such tetracycline, chloramphenicol and nalidxic acid. The results have showed that from 10 isolates of A. hydrophila tested, five isolates were resistant, two isolates were intermediate and three isolates were sensitive to 2

tetracycline and chloramphenicol. While one isolate was resistant, six isolates were intermediate and two isolates were sensitive to nalidxic acid. From the above findings, that majority of A. hydrophila strains were resistance to antibiotics due to the development of bacterial antibiotic resistance.

Vaccines are also well-known to control both bacterial and viral diseases. Unfortunately, vaccination is an expensive, time consuming and complicated process (Taylor, 2012). Moreover, killed vaccines must be inoculated via injection, thus ineffective for commercial application; and live vaccines are known to have possible reversion to virulence (Trust, 1986). Therefore, the use of probiotic bacteria has been suggested to become an alternative method to prevent and control various diseases in aquaculture while improving growth and survival (Chiu et al., 2010; Sun et al., 2010; Son et al., 2009). Moreover, probiotics can be applied at larval and fry stages, where vaccination cannot (Mohapatra et al., 2012). UPM 1.3 Objectives

Bacillus subtilis strain G1 used in this study was isolated by Zokaeifar et al. (2012a) from fermented pickles. It has shown to improve the growth of juvenile marine shrimp (Litopenaeus vannamei) and increases the survivability of the shrimp from vibriosis. Therefore, this study was conducted on freshwater species against freshwater pathogens. The aims of this study were;

a) To investigate the effect of Bacillus subtilis G1 on the growth performance and body composition of H. nemurus fingerlings

b) To investigate the effect of Bacillus subtilis G1 on disease resistance towards A. hydrophila infection on H. nemurus fingerlings.

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